Photosynthesis

Question 1

What entropy does life require?

Answer 1

Negative entropy

Question 2

What is ∆p?
What does it drive the formation of?

Answer 2

∆p – Electrochemical proton gradient/Proton motive force (pmf)

Drives the formation of ATP

Question 3

Explain the Jagendorf acid-bath experiment
(refer to image on notes)

Answer 3

Thylakoids are incubated in a low pH medium in the dark containing a weak permeable acid

The acid will diffuse across the membrane and dissociate inside to equilibrate the pH to 4 inside

It is then rapidly transferred to a high pH medium, and a transient ∆p is created, allowing ATP synthesis if ADP and Pi are added

Question 4

Give evidence for chemiosmosis (hint: uncoupler)

Answer 4

If the proton gradient formed by electron transport is needed to generate ATP, then its abolition should inhibit ATP formation

The proton gradient can be abolished by an un-coupler; A molecule which facilitates diffusion of protons across the normally impermeable membrane
- ATP synthesis was abolished; Electron transport and ATP synthesis were now uncoupled

A light-driven proton pump ‘bacteriorhodopsin’ could, when reconstituted in lipid vesicles with ATP synthase, drive the production of ATP

Question 5

What are NADH and FADH2 and what happens to them during respiration?

Answer 5

They are electron carriers

They are reduced in glycolysis, the link reaction and the citric acid cycle, and then oxidised in the mitochondria to make ATP

Question 6

What is reductant-powered electron transfer coupled to?
What does this generate?

Answer 6

Proton transfer across a membrane

This generates electrochemical proton gradient (∆p)

Question 7

Where does photosynthetic electron transport occur?
Where does CO2 fixation occur?

Answer 7

Thylakoid membrane

Stroma

Question 8

What does light energy do to electrons when absorbed?
What is done with the free energy that is released?

Answer 8

Excites electrons; Raises the redox potential from positive to negative

Free energy released used to generate ∆p for ATP synthesis

Question 9

What is done with the free energy released when electrons flow from a negative to positive redox potential? (give an example)
What is this called?

Answer 9

Free energy released can be used to do work e.g. move protons from an area of low concentration (matrix) to an area of high concentration (MIS)

This is called chemiosmotic coupling

Question 10

Similarities and Differences of Respiration and Photosynthesis?

Answer 10

Both involve electrons being transferred down gradient of redox potential with free energy released used to power formation of ∆p for ATP synthesis

Respiration uses electrons from reductants
Photosynthesis uses electrons energised by light

Question 11

What is the structure of Chlorophyll? (Similarities with haem?)

Answer 11

It formed of 2 parts:
- Tetrapyrrole ring; Similar to haem but it coordinates an Mg2+ ion, not an Fe2+
- Hydrophobic phytyl tail

Question 12

What system in the tetrapyrrole ring is responsible for light absorption?
What happens when chlorophyll absorbs light?

Answer 12

Conjugated pi-electron system

When chlorophyll absorbs light an electron in this region is promoted to a higher energy level

Question 13

What complexes do light driven reactions occur in?

What do they result in? (hint: electron transfer; by-products?)

Answer 13

PSII and PSI

They result in electron transfer via a chain of acceptors from water to NADP+, with oxygen formed as a by-product

Question 14

How do the reaction centres use light energy?

Answer 14

To drive energetically unfavourable reactions (+∆G) that transfer an electron from a donor with a positive redox potential (water) to an acceptor with a more negative redox potential (NADP+) i.e. uphill

Question 15

+∆G vs -∆G

Is a negative to positive redox potential favourable or unfavourable

Answer 15

+∆G - Unfavourable reactions
-∆G - Favourable reactions

Favourable (-∆G)

Question 16

What is the typical redox potential for acceptors and donors

Answer 16

Donors - Negative redox potential
Acceptors - Positive redox potential

Question 17

What is the structure of a photosystem?

Answer 17

Antenna complex is formed of hundreds of chlorophylls which transfer absorbed light energy to the special pair chlorophylls of the reaction centre that are redox active

Question 18

Why are antenna systems needed?

Answer 18

The antenna increases RC excitation rate by 2 orders of magnitude
The antenna acts to capture and concentrate light energy

Question 19

What does variation in the length of the conjugated pi-electron affect?

Answer 19

It affects the wavelengths of light absorbed by each pigment

Question 20

Why are multiple pigment types combined in the antenna?

Answer 20

To broaden the spectral cross-section of light energy that is absorbed and transferred to the reaction centre chlorophylls

Question 21

How are antenna proteins and pigments linked?
Where does the complex sit and why?

Answer 21

Antenna proteins non-covalently bind pigments at high concentrations to ensure efficient light absorption

This complex sits in the membrane and is extremely hydrophobic

Question 22

How are PSII and Light Harvesting Complex II (LHCII) linked?
How is the system modular?

Answer 22

They form a dimeric super complex

Modular system as there is more LHCII when growing under low light, and less LHCII when growing under high light

Question 23

How are PSI and Light Harvesting Complex I (LHCI) linked?
How is the system modular?

Answer 23

They form a monomeric super complex

Modular system as there is more LHCI when growing under low light, and less LHCI when growing under high light

Question 24

What photons do molecules absorb?

What is fluorescence and which state does it occur from?

Answer 24

They only absorb photons with energy equal to the energy gap between electron orbitals

Fluorescence is when photons are re-emitted; This always occurs from the lowest excited state

Question 25

How many major excited states does chlorophyll have? Which states do red and blue photons match?

Answer 25

Chlorophyll has 2 major excited states Red photons match the S0->S1 (first excited state) gap Blue photons have more energy than red photons (shorter wavelength) and so they excite electrons to the S2 state

Question 26

What happens to an electron excited by a blue photon?

Answer 26

Promoted to S2 state Rapidly loses some energy through vibrational relaxation Drops to S1 state through internal conversion (heat) Further vibrational relaxation reduces electrons energy to lowest energy level of the S1 state

Question 27

What then happens to the photon in the S1 state? (hint: 2 ways)

Answer 27

Internal conversion from S1 to S0 occurs, but more slowly than S2 to S1 As it is slower, fluorescence (photon emission) competes as an alternative channel of de-excitation

Question 28

Why is internal conversion from S1 to S0 slower than that for S2 to S1?

Answer 28

S1 to S0 is slower as the electron is closer to the nucleus and so is more stable

Question 29

What happens to S1 electrons between adjacent chlorophylls?

Answer 29

Fluorescence and internal conversion are so slow that FRET can compete

Question 30

What is FRET?

Answer 30

Forster Resonance Energy Transfer This occurs if 2 chlorophylls are in close proximity, with excited state energy levels that overlap Essentially transfers excitation energy from an electron on one chlorophyll to another

Question 31

What is resonance?

Answer 31

When the donor emission and acceptor absorption spectra overlap, FRET can occur between them in a distance (R0) dependent fashion

Question 32

Explain FRET efficiency (hint: distance)

Answer 32

The efficiency of FRET varies with the 6th power of the distance i.e. If the distance between 2 molecules doubles, the FRET transfer time increases by 64 times FRET is only efficient over short distances (<7nm)

Question 33

How is each pigment binding site in LHCII different? Effect of this? (hint: spectra and flow)

Answer 33

They all vary in there excited state properties: - Energy - Spectra - Excited state lifetime This diversity in binding sites energies broadens spectral cross-section further and creates directionality in energy flow

Question 34

How is directionality achieved through the range of binding sites? What happens in the RC?

Answer 34

Chlorophylls closer to the reaction centre (RC) have excited state at lower energies than those further out in the antenna So excitation energy cascades downhill towards the RC by FRET, where it is ‘trapped’ as an electron transfer reaction

Question 35

Name for special pair in PSII?

Answer 35

P680

Question 36

What redox reactions is PSII involved in?

Answer 36

Oxidising H2O to O2 Reducing PQ to PQH2

Question 37

Equation for energy of 1 mole of photons? Meaning of each term?

Answer 37

ΔG = (Nhc)/wavelength of light N - Avogadro's number h - Planck's constant c - Speed of light in vacuum

Question 38

∆E0’ equation?

Answer 38

∆E0’ = E0’(acceptor) – E0’(donor)

Question 39

What co-factor is key for PSII electron transfer?

Answer 39

Manganese cluster

Question 40

How many turnovers of PSII electron transfer is required for 1 PQH2? How many for 1 O2?

Answer 40

2 turnovers for 1 PQH2 4 turnovers for 1 O2

Question 41

Process of PSII electron transfer reactions (refer to images on notes) What branch(es) does charge separation occur down in PSII?

Answer 41

Just down branch A

Question 42

What happens to electrons in chlorophyll when excited? What does the Mg2+ ion do?

Answer 42

Upon excitation, the electron is lost from the tetrapyrrole ring in the RC chlorophyll rather than from the Mg2+ ion which is redox inactive The resulting hole/electron is delocalised over the tetrapyrrole ring In chlorophyll the Mg2+ ion tunes the absorption spectrum of the molecule and provides a coordination site for interaction with protein

Question 43

FRET between antenna and RC; Which direction?

Answer 43

FRET only occurs from antenna to RC No transfer from RC to antenna

Question 44

How is plastoquinone converted to plastoquinol? What happens after the conversion?

Answer 44

2 e- reduce the 2 C=O carbons on plastoquinone from +2 to +1 oxidation state, forming 2 OH- 2 H+ are taken up from the stroma to form plastoquinol Once PQH2 is formed at the QB site, it is exchanged for PQ

Question 45

How is the manganese cluster changed and then restored to its original state?

Answer 45

Manganese ions in the cluster are progressively oxidised It is restored to its original state by electrons from H2O

Question 46

How is charge separation stabilised?

Answer 46

A chain of electron acceptors energetically ‘downhill’ from P680* ensures e- and hole (+) are rapidly separated spatially

Question 47

2 key molecules in PSI? What enzyme catalyses their reaction? (hint: tells us which is reduced)

Answer 47

Ferredoxin and Plastocyanin Ferredoxin Oxidoreductase

Question 48

Which molecule is oxidised and which is reduced by PSI?

Answer 48

PSI oxidises plastocyanin and reduces ferredoxin

Question 49

Name for special pair in PSI? What branch(es) does charge separation occur down in PSI?

Answer 49

P700 Branch A or B

Question 50

Process of electron transfer in PSI (see image on notes)

Question 51

How is the electron hole filled in PSI?

Answer 51

P700+ is re-reduced by an e- from plastocyanin

Question 52

What are Ferredoxin and Plastocyanin?

Answer 52

They are both small soluble electron transfer proteins

Question 53

Key feature of Plastocyanin? (hint: ion) What happens to this feature? (hint: Cb6f)

Answer 53

A copper ion is bound at its active site; This acts as the electron carrier The copper ion is oxidised from Cu+ to Cu2+ by PSI, and reduced back by cytochrome b6f

Question 54

What does Ferredoxin bind at its active site?

Answer 54

Binds a 2Fe-2S cluster at its active site that acts as the electron carrier

Question 55

What does Marcus theory say about the rate of ET reaction and direct charge recombination?

Answer 55

ET reaction rate decreases in the 'inverted region' as large driving forces between redox couples slows ET rates Charge recombination falls in the inverted region and a slow ET rate - More likely for electron to travel along a branch than recombine with the hole

Question 56

Structure of Cytochrome b6f? What does it bind per monomer?

Answer 56

Dimer - 2 x PQ - 1 x carotenoid - 1 x chlorophyll - 4 x haems - 1 x 2Fe2S cluster

Question 57

Co-factors in cytochrome b6f? What are they linked to? What cluster is at the centre and what is it coordinated by?

Answer 57

2 c-type haems 2 b-type haems c-type haems are linked to protein b-type haems are linked to central Fe ion Iron-sulphur cluster (2Fe2S) Coordinated by histidine and cysteine

Question 58

Process of bifurcated electron transfer (2 steps) (hint: how Cu2+ is reduced) (see images on notes)

Answer 58

Step 1: - PQH2 is oxidised - 2 protons are deposited into the lumen and the 2 electrons follow different paths (bifurcate) - 1 electron reduces Pc, and 1 electron is stored in haem cn Step 2: - Another PQH2 is oxidised - 2 protons are again deposited into the lumen and the 2 electrons follow different paths (bifurcate) - 1 electron reduces a second Pc, and 2 electrons reduce the PQ molecule to PQ-2, which binds 2 protons from stroma to become PQH2

Question 59

What happens during the proton motive Q-cycle? (hint: recycling)

Answer 59

Recycling of 1 of the 2 electrons from each PQH2, doubles the number of protons transferred by the complex per PQH2 oxidised, boosting its contribution to the proton motive force (∆p)

Question 60

What does NADP+ reduction by FNR require? What molecule has this function? (hint: storage)

Answer 60

Requires 2 electrons and 1 proton and a place to store 1 electron from ferredoxin, while the FNR waits for the second to arrive Flavin Adenine Dinucleotide (FAD) of FNR has this function

Question 60

What complex is the 'end of the line' for photosynthetic electron? What happens here?

Answer 60

Ferredoxin-NADP+ reductase (FNR) NADP+ is reduced to NADPH

Question 61

The calvin cycle requires 1.5ATP per NADPH, but only 1.28 ATPs are formed per NADPH in a chloroplast How is the imbalance in ATP/NADPH ratio corrected?

Answer 61

Cyclic electron transport

Question 62

What does cyclic electron transport involve? (hint: returning electrons) What complex is involved in this pathway? (what enzyme does this act as?) How does it contribute to ATP formation and correct the imbalance?

Answer 62

Involves returning electrons from Ferredoxin to the PQ pool Photosynthetic complex I; Acts as ferredoxin-plastoquinone reductase Contributes to pmf, and therefore ATP formation; No NADPH synthesis

Question 63

What enzyme does photosynthetic complex I act as? What do the 4Fe4S clusters do?

Answer 63

Ferredoxin-plastoquinone reductase The 4Fe4S clusters shuttle electrons from Fd to PQ to make PQH2

Question 64

What is done with the free energy released in the PQ -> PQH2 reaction?

Answer 64

Powers movement of 4H+ across membrane; Contributes to pmf

Question 65

Parts of ATP synthase in F-type ATP synthesis?

Answer 65

F1 rotor is where ADP+Pi -> ATP occurs Clamp holds 2 parts of the enzyme together

Question 66

Sets of subunits of ATP synthase (5 sets)

Answer 66

a-subunit c-subunit γ-subunit α and β subunits b2 and δ subunits

Question 67

What do the a and c subunits do?

Answer 67

a-subunit - Half channels; Lets protons in on one side, and out on the other side c-subunit - Proton binding subunits; Number of c subunits varies

Question 68

What does the γ-subunit do? (2 functions)

Answer 68

Central stalk that transmits the torque from the c-subunit to the head part of the protein Also cycles β-subunit between open, loose and tight states

Question 69

What do α and β, and b2 and δ subunits do? (hint: α and β have different functions but link)

Answer 69

α and β subunits - 3 of each form the F1 head - β are catalytically active; α has a structural role, and helps β form the binding site for ADP, Pi and ATP b2 and δ subunits - Form the ‘clamp’, bridging the F1 and F0 domains, ensuring the 2 parts stay coupled

Question 70

How do protons bind to c-subunits? What happens in a hydrophobic environment? (hint: pKa)

Answer 70

Each c-subunit has a glutamate residue with a carboxy group (negative) to bind protons In hydrophobic environment, the pKa for the carboxy group is high; This means the group will be protonated at a more basic pH (low pKa means it is protonated at a more acidic pH)

Question 71

What residues are present on the a-subunit? What groups do they have and what do they bind?

Answer 71

Arginine residues are present, which can bind protons with an amine group

Question 72

What happens when glutamate and arginine residues are in close proximity? (hint: COO- and pKa)

Answer 72

COO- is stabilised by the positive charge of arginine This causes the carboxy groups pKa to drop and become deprotonated and the proton exits the half channel

Question 73

What happens to deprotonated glutamate? (hint: its on side of high H+ conc.) What does this cause to happen and why?

Answer 73

Deprotonated glutamate has low pKa and is on side of high H+, so it can still be protonated The protonation causes the c-ring to rotate due to the attraction between the positive arginine and the negative glutamate

Question 74

What happens when the γ-subunit rotates? What are the 3 states?

Answer 74

β-subunit conformation changes between 3 states - O state - L state - T state

Question 75

What happens during the 3 states? (hint: involves ATP, ADP and Pi)

Answer 75

O state - ATP released L state - ADP + Pi is bound T state - ADP + Pi is converted to ATP

Question 76

How many ATP from one c-ring rotation? How many protons required for one complete rotation of γ-subunit?

Answer 76

3 ATP per c-ring rotation 1 proton required for every c-subunit there is, to then produce a γ-subunit

Question 77

With the same pmf, would a smaller or larger c-ring be faster at ATP synthesis?

Answer 77

Faster with smaller c-ring

Question 78

With different pmfs, would it be easier to produce ATP with a smaller or larger c-ring? Why?

Answer 78

Easier to produce ATP with a larger c-ring This is because it requires less pmf to do the same work compared to a smaller c-ring The larger the c-ring, the larger the pmf multiplier

Question 79

Summary: Out of a smaller or larger c-ring, which is more optimised for rapid synthesis, and which is more optimised for low energy inputs What do they both sacrifice?

Answer 79

Smaller ring is optimised for rapid synthesis sacrificing efficiency Larger ring is optimised for low energy inputs, sacrificing speed

Question 80

Is ATP synthase reversible? What does the direction depend on?

Answer 80

It is reversible The direction depends on thermodynamic balance between ∆p and ∆G-ATP

Question 81

What are the 3 parts of the calvin cycle?

Answer 81

Carboxylation Reduction Regeneration

Question 82

How many molecules of ATP and NADPH are required to reduce 3 CO2, for every complete turn of the cycle? How many molecules of glycerdaldehyde-3-phosphate are produced?

Answer 82

9 ATP 6 NADPH 6 glyceraldehyde-3-phosphate is produced

Question 83

What occurs during carboxylation? Enzymes involved in catalysis?

Answer 83

CO2 is combined with ribulose-1,5-bisphosphate; Catalysed by RuBisCO Forms an unstable 6 carbon intermediate that splits into 2 molecules of 3-phosphoglycerate

Question 84

Characteristics of RuBisCO? Subunits? (2 types)

Answer 84

Slow enzyme Low affinity for CO2 High concentrations are needed to match potential supply of ATP and NADPH 8 large catalytic and 8 small regulatory subunits

Question 85

What occurs during reduction? (2 steps) What is the end product and its major fate?

Answer 85

First step involves 3-phosphoglycerate is phosphorylated to form 1,3-bisphosphoglycerate using ATP from light reactions The second step involves reduction of 1,3bisphosphoglycerate using NADPH to form glyceraldehyde 3-phosphate (GAP) GAP is the output and is used in sucrose synthesis

Question 86

What occurs during regeneration? How many molecules of GAP per ribulose-1,5-bisphosphate and CO2? What is done with the majority of GAP? Final step?

Answer 86

6 GAP for every: - 3 ribulose-1,5-bisphosphate - 3 CO2 5/6 GAP molecules are used to regenerate ribulose 5-bisphosphate Ribulose 5-phosphate is phosphorylated into ribulose 1,5-bisphosphate using ATP from light reactions

Question 87

How does the activity of the light reactions link to and regulate dark reaction enzymes? (2 ways) (hint: thioredoxin and ∆p)

Answer 87

∆p is formed across the thylakoid membrane This increases the pH and the [Mg2+] in the stroma - H+ influx into the lumen is balanced by Mg2+ efflux to the stroma Ferredoxin and NADP+ are reduced, changing redox state of the stroma, which is sensed by the thioredoxin Thioredoxin regulates several calvin cycle enzymes

Question 88

How is RuBisCO regulated? (hint: light reactions)

Answer 88

RuBisCO active site contains a lysine which reacts with CO2 to form a carbamate anion that can bind to Mg2+ Mg2+ is provided by the light reactions Mg2+ is essential for the RuBisCO to function as it activates ribulose 1,5-bisphosphate